Airway epithelial cells are active participants in the immune defense of the lung. As part of the mucosal immune system, it seems likely that airway cells should share major signaling pathways with "professional" immune cells. T and B cells generate Ca2+ fluxes in response to antigen as a major mechanism to activate gene expression. Mucosal epithelial cells also respond to bacterial ligands with the activation of this common intracellular messenger. In airway epithelial cells Ca2+ dependent signaling pathways are activated by Gram positive and Gram negative bacteria to stimulate expression of cytokines and particularly the chemokine IL- 8. Immediately upon contact by either S. aureus or P.aeruginosa, epithelial cells release Ca2+ from intracellular stores. These Ca2+ fluxes are sufficient to induce downstream signaling, NF-kappaB activation and IL-8 transcription. We postulate that a spatially organized receptor complex, which includes glycolipids and toll like receptors is mobilized to the apical surface of airway cells in response to specific bacterial ligands. In the experiments proposed, we will identify the components of this lipid raft signaling complex that are important in epithelial activation. The generation of Ca2+ fluxes appears to be a common central theme in the activation of immune signaling. We will establish how epithelial cells generate Ca2+ fluxes, the involvement of PI3K and PLCgamma in response to bacterial ligands, as well as the participation of Src, Ras, Btk, and CaMK's. Bacterial signaling is mediated by toll like receptors and particularly TLR2 in airway cells. We will determine how the activation of TLR2 generates Ca2+ signaling, which kinases are involved, and how they are co-localized at the apical pole of airway cells. This will be accomplished by studying signaling in both TLR2 null and caveolin-1 null mice and in epithelial cell lines derived from these animals. We will delineate the major components of this signaling cascade, identifying which Ca2+-dependent kinases, phosphatases and transcription factors including NFAT and CREB as well as NF-kappaB are involved. Such Ca2+ activated responses are likely to be important in airway inflammation induced by chronic bacterial infection, as in airway diseases such as cystic fibrosis. Exactly how CFTR dysfunction affects Ca2+ signaling will be explored. CFTR mutations may influence basal Ca2+ levels in the cell. Repeated apical exposure to bacteria may cause receptor clustering and increase the efficiency of epithelial signaling. By identifying the components of these pathways it should be possible to target therapy to ameliorate airway inflammation in diseases such as cystic fibrosis.